title: FACT is recruited to the +1 nucleosome of transcribed genes and spreads in a Chd1-dependent manner [MNase-ChIP-Seq]

first: Francois

last: Robert

state: QC

address: 110 av des Pins Ouest

institute: IRCM

summary: The histone chaperone FACT occupies transcribed regions where it plays prominent roles in maintaining chromatin integrity and preserving epigenetic information. How it is targeted to transcribed regions, however, remains unclear. Proposed models for how FACT finds its way to transcriptionally active chromatin include docking on the RNA polymerase II (RNAPII) C-terminal domain (CTD), recruitment by elongation factors, recognition of modified histone tails and binding partially disassembled nucleosomes. Here, we systematically tested these and other scenarios in Saccharomyces cerevisiae and found that FACT binds transcribed chromatin, not RNAPII. Through a combination of experimental and mathematical modeling evidence, we propose that FACT recognizes the +1 nucleosome, as it is partially unwrapped by the engaging RNAPII, and spreads to downstream nucleosomes aided by the chromatin remodeler Chd1. Our work clarifies how FACT interacts with genes, suggests a processive mechanism for FACT function, and provides a framework to further dissect the molecular mechanisms of transcription-coupled histone chaperoning.

overall_design: Paired-end sequencing of immunoprecipitated micrococcal nuclease-treated chromatin  (MNase-ChIP-seq) of both FACT subunits (Spt16 and Pob3) in wild type and chd1Δ cells, with corresponding Inputs. The data also includes MNase-ChIP-seq for Chd1 in wild type and chd1K407R cells, with corresponding Inputs. All data are from two independent biological replicates.

first: Célia

last: Jeronimo

first: Andrew

last: Angel

first: Christian

last: Poitras

first: Pierre

last: Collin

first: Jane

last: Mellor

first: François


title: Transcriptional profiling of Caenorhabditis elegans in control (20°C) and heat shock (35°C) of N2 (WT) and miR-85(n4117) animals

status: Public on Jul 26 2021

city: La Jolla

first: Delaney

last: Pagliuso

state: CA

address: 9500 Gilman Dr

department: Biological Sciences

country: USA

institute: University of California, San Diego

type: Expression profiling by high throughput sequencing

summary: Transcriptional profiling of N2 (WT) and miR-85(m4117) Caenorhabditis elegans at larval stage 4 (L4) compared at either control temperature (20°C) or after 3hr HS (35°C).

overall_design: 12 total samples; 3 biological replicates of N2 (WT) and miR-85(n4117) maintained at control (20°C) temperatures and heat shock treated for 3 hours (35°C)

middle: C

first: Amy

middle: E

last: Pasquinelli


title: Ablation of Histone Demethylase KDM5B in Melanoma Augments Anti-Tumor Immunity through Upregulation of Retroelements [ATAC-seq]

status: Public on Jul 25 2021

city: New Haven

first: Qin

last: Yan

email: qin.yan@yale.edu, craig.wilen@yale.edu

address: 310 Cedar St.

department: Pathology

institute: Yale University

type: Genome binding/occupancy profiling by high throughput sequencing

summary:
Purpose: Advanced melanoma patients have poor prognosis. Although immune checkpoint blockade has revolutionized treatment for melanoma patients, majority of patients do not respond. The goal of this research is to evaluate whether epigenetic therapy targeting KDM5B could overcome resistance to immunotherapy.
Methods: ATAC-Seq analysis of mouse melanoma cell line YUMMER1.7.
Results: We identified that KDM5B directly bind to retreoelements to repress their expression.
Conclusions: Our work characterized ablation of histone demethylase KDM5B in melanoma augments anti-tumor immunity through Upregulation of retroelements.

overall_design: ATAC-seq analysis in mouse melanoma cell lines

first: Shang-Min

last: Zhang

first: Wesley

middle: L

last: Cai


title: Lentiviral-based mutagenesis to identify mutations that confer resistance to anti-cancer drugs [lenti]

city: Dallas

first: Ralf

last: Kittler

state: Texas

address: 5323 Harry Hines Blvd

institute: University of Texas Southwestern Medical Center

summary: Identifying resistance mutations in a drug target provides crucial information. Lentiviral transduction creates multiple types of mutations due to the error-prone nature of the HIV-1 reverse transcriptase (RT) and we show this property can be leveraged to identify mutations that confer resistance to targeted anti-cancer drugs, a technique we term “LentiMutate”. First, we improved LentiMutate by making the lentiviral RT more error-prone. Next, we applied this technique to two anti-cancer drugs, imatinib and AMG 510. We find novel deletions in BCR-ABL that confer resistance to BCR-ABL inhibitors and point mutations in the AMG 510 binding pocket or oncogenic non-G12C mutations, in KRAS-G12C or wild-type KRAS, respectively, that confer resistance to AMG 510. LentiMutate may prove highly valuable to clinical and preclinical cancer drug development

overall_design: The overall design of all these experiments is a technique known as LentiMutate. Here a cDNA of a drug target is packaged into a lentiviral particle, which then infects cells, generates mutations at a low frequency, and then the cells are treated with an anti-cancer drug that targets the cDNA. Cells that have mutations in the cDNA that cause resistance to the drug are able to survive drug treatment, and the cDNA in these cells is sequenced and compared to the parental population to identify drug resistnace mutations (since drug resistance mutations will be enriched in this surviving population)

first: Paul

last: Yenerall

first: Rahul

middle: K

last: Kollipara

first: Kimberly

last: Avila

first: Michael

last: Peyton

first: Christopher

last: Eide

first: Yan

last: Liu

first: Kenneth

last: Westover

first: Brian

last: Druker

first: John

middle: D

last: Minna


title: Lentiviral-based mutagenesis to identify mutations that confer resistance to anti-cancer drugs [umi]

overall_design: HCC827 cells were infected with lentiviruses harboring the EGFRdel746-750 cDNA packaged using either the M- or WT-reverse transcriptase (RT). Lentiviral integrants in these cells were PCR amplified using primers with unique molecular identifiers to determine the mutation rate in the EGFR cargo when packaged using the M-RT or WT-RT.


title: Early-life antibiotic-driven dysbiosis in mice is associated with impaired metabolism and immunity in later life and reduced lifespan.

city: Adelaide

first: Feargal

middle: J

last: Ryan

state: Adelaide

address: North Terrace

country: Australia

institute: SAHMRI

summary: RNA-Seq of livers from aged mice exposed to antibiotics

overall_design: 16 liver samples from aged mice from antibiotic exposed and no antibiotics controls, sequenced to a mean depth of 60 million 2x100bp reads on an Illumina NovaSeq

first: David

last: Lynn


title: The polar flagellar transcriptional regulatory network in Vibrio campbellii deviates from known Vibrio species

city: Bloomington

first: Julia

last: van Kessel

state: IN

address: 1001 E 3rd Street

department: Biology

institute: Indiana University

summary: Vibrio campbellii is a gram-negative bacterial pathogen that is both free-living and a pathogen of marine organisms and exhibits swimming motility via a single, polar flagellum. Swimming motility is a critical virulence factor in V. campbellii pathogenesis, and disruption of the flagellar motor significantly decreases host mortality. However, while V. campbelli encodes homologs of flagellar and chemotaxis genes conserved by other members of the Vibrionaceae, the regulatory network governing these genes have not been explored. We systematically deleted all 63 known flagellar and chemotaxis genes in V. campbellii and examined their effects on motility compared to their homologs in other Vibrios. We specifically focused on assessing the roles of the core flagellar regulators of the flagellar regulatory hierarchy established in other Vibrios: rpoN, flrA, flrC, and fliA.  Although V. campbellii transcription of flagellar and chemotaxis genes is governed by a multi-tiered regulatory hierarchy similar to other Vibrios, we observed two critical differences: the σ54-dependent regulator FlrA is dispensable for motility, and Class II gene expression is independent of σ54 regulation. Our genetic and phenotypic dissection of the V. campbellii flagellar regulatory network highlights the differences that have evolved in flagellar regulation across the Vibrionaceae.

overall_design: RNA-Seq of cultures grown to OD600=1.0

first: Blake

last: Petersen

middle: S

first: Ram

last: Podicheti

first: Ying-Po

last: Yang

first: Chelsea

middle: A

last: Simpson

first: Kai

last: Papenfort

first: Chris

last: Hemmerich

first: Douglas

middle: B

last: Rusch


title: Ablation of Histone Demethylase KDM5B in Melanoma Augments Anti-Tumor Immunity through Upregulation of Retroelements [ChIP-Seq]

first: Qin

last: Yan

summary:
Purpose: Advanced melanoma patients have poor prognosis. Although immune checkpoint blockade has revolutionized treatment for melanoma patients, majority of patients do not respond. The goal of this research is to evaluate whether epigenetic therapy targeting KDM5B could overcome resistance to immunotherapy.
Methods:ChIP-Seq analysis of KDM5B binding on retroelements in mouse melanoma cell line 1445.
Results: We identified that KDM5B directly bind to retreoelements to repress their expression.
Conclusions: Our work characterized ablation of histone demethylase KDM5B in melanoma augments anti-tumor immunity through Upregulation of retroelements.

overall_design: ChIP-seq analysis of KDM5B in mouse melanoma cell lines; ChIP-qPCR were conducted to verify KDM5B's binding to retroelement loci.

first: Shang-Min

last: Zhang

first: Wesley

last: Cai


title: FACT is recruited to the +1 nucleosome of transcribed genes and spreads in a Chd1-dependent manner

city: Montreal

first: Francois

last: Robert

country: Canada

type: Genome binding/occupancy profiling by array

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: New-Onset IgG Autoantibodies in Hospitalized Patients with COVID-19 [Paired Cytokine]

status: Public on Jul 24 2021

city: Stanford

first: Allan

last: Feng

address: 269 Campus Dr

department: Imm/Rheum

institute: Stanford School of Medicine

summary: We developed three different protein arrays to measure IgG autoantibodies associated with Connective Tissue Diseases (CTDs), Anti-Cytokine Antibodies (ACA), and anti-viral antibody responses in 147 hospitalized COVID-19 patients in three different centers.

platform_organism:
Escherichia coli
Triticum aestivum
Spodoptera frugiperda
Homo sapiens
Sus scrofa
Bos taurus
Capra aegagrus
Cricetulus griseus

overall_design: Protein arrays were constructed by coupling antigens to carboxylated magnetic beads. Arrays consisting of conjugated beads were then used to probe hospitalized COVID-19 patient serum samples, healthy control samples, and autoimmune prototype samples  to identify autoantibodies. Samples were run in duplicate except one sample (UP67 D7) which was run in quadruplicate. for A Luminex FLEXMAP 3D® system was used to quantitatively measure autoantibody reactivity in each sample in units of mean flourescent intensity (MFI). Positive antibody hits were statistically identified by a 5 standard deviation cutoff calculated from the MFI of healthy controls.

first: Sarah

last: Chang

first: Wenzhao

last: Meng

first: Sokratis

last: Apostolidis

first: Chrysanthi

last: Skevaki

first: Eline

middle: T

last: Luning Prak

first: Paul

last: Utz


title: Genes altered by miR-766-5p in two cancer cell lines

city: Tokyo

first: Yasuyuki

last: Gen

address: 1-5-45 Yushima, Bunkyo-ku, Tokyo

country: Japan

institute: Tokyo Medical and Dental University

summary: To identify genes affected by miR-766-5p overexpression, we transfected with 10nmol of miR-766-5p or miR-negative control (NC) in HCT116-/-, or MIAPaCa2 cells. After 2 days, RNA was extracted, and then expression analysis was performed using agilent microarray.

overall_design: Expression microarray with miR-766-5p or miR-NC transfected HCT116-/-, or MIAPaCa2 cells were performed in duplicate.

first: Johji

last: Inazawa


title: New-Onset IgG Autoantibodies in Hospitalized Patients with COVID-19 [Unpaired Autoantigen V2]


title: New-Onset IgG Autoantibodies in Hospitalized Patients with COVID-19 [Autoantigen V2]


title: Single-cell  transcriptomic profiles of CD45-negative cells from WT (C57BL/6), antibiotics non-treated Mx1Cre-Adam10f/f and antibiotics treated Mx1Cre-Adam10f/f mice

city: Bethesda

first: Keisuke

middle: Chris

last: Nagao

state: Maryland

address: 9000 Rockville Pike, Bldg 10, Rm12N240B

institute: National Institute of Arthritis and Musculoskeletal and Skin Diseases

summary: Induced abltion of Adam10 in Mx1-Cre x Adam10f/f mice results in T cell-independent Inflammatory alopecia. To determine if dysbiosis is a prerequisite for the development of alopecia, Mx1-Cre x Adam10f/f mice were treated orally with an antibiotic cocktail pre- and post-induction. 30 days after poly(I:C) injection, mouse skin was dissociated and were sorted for CD45-negative cells which were subjected to single-cell RNA sequencing analysis.We found that the ADAM10-Notch signaling axis in type I/III interferon-responsive upper hair follicles (HF) was crucial for the expression of genes that regulated skin microbiota and protected HFs and its stem cell niche from inflammatory destruction.

overall_design: Single-cell transcriptomic profiles of CD45-negative cells sorted from WT, antibiotics non-treated Mx1Cre-Adam10f/f  and antibiotics treated Mx1Cre-Adam10f/f mice 30 days after poly(I:C) injetion.

first: Keiko

last: Sakamoto

first: Seon-Pil

last: Jin


title: New-Onset IgG Autoantibodies in Hospitalized Patients with COVID-19 [Autoantigen V1]


title: New-Onset IgG Autoantibodies in Hospitalized Patients with COVID-19

sample_organism:
Homo sapiens
synthetic construct


title: Single-cell  transcriptomic profiles of CD45-negative cells from Mx1Cre-Rbpjf/f mice

summary: Induced abltion of Adam10 in Mx1-Cre x Adam10f/f mice results in T cell-independent Inflammatory alopecia. To determine if the Notch signaling pathway was downstream of Adam10,  Mx1-Cre x Rbpjf/f mice were generated. After poly(I:C) injection, mouse skin was dissociated and were sorted for CD45-negative cells which were subjected to single-cell RNA sequencing analysis.We found that the ADAM10-Notch signaling axis in type I/III interferon-responsive upper hair follicles (HF) was crucial for the expression of genes that regulated skin microbiota and protected HFs and its stem cell niche from inflammatory destruction.

overall_design: Single-cell transcriptomic profiles of CD45-negative cells sorted from Mx1Cre-Rbpjf/f mice 30 days after poly(I:C) injetion.


title: Ablation of Histone Demethylase KDM5B in Melanoma Augments Anti-Tumor Immunity through Upregulation of Retroelements [RNA-Seq YUMMER1.7-Kdm5b KO]

summary:
Purpose: Advanced melanoma patients have poor prognosis. Although immune checkpoint blockade has revolutionized treatment for melanoma patients, majority of patients do not respond. The goal of this research is to evaluate whether epigenetic therapy targeting KDM5B could overcome resistance to immunotherapy.
Methods:RNA-Seq analysis of KDM5B KO mouse melanoma cell lines compared to control cells to evaluate whether KDM5B depletion induces activation of retroelements, which subsequently activates type I interferon responses. Mouse studies were conducted to evaluate whether anti-tumor immunity induced by KDM5B loss could overcome immunotherapy resistance.
Results: We identified that KDM5B depletion derepress retroelement expression, which subsequently activates type I interferon response and enhances anti-tumor immunity. Loss of KDM5B could overcome resistance to anti-PD-1 immunotherapy.
Conclusions: Our work characterized ablation of histone demethylase KDM5B in melanoma augments anti-tumor immunity through Upregulation of retroelements.

overall_design: RNA-seq analysis of KDM5B KO vs Control mouse melanoma cell lines; in vitro and in vivo functional study; molecular mechanism study


title: Non-redundant functions of H2A.Z.1 and H2A.Z.2 in chromosome segregation and cell cycle progression

city: Uxbridge

first: Paola

last: Vagnarelli

address: Kingstone Lane

country: United Kingdom

institute: Brunel University London

summary: H2A.Z is a H2A-type histone variant essential for many aspects of cell biology, ranging from gene expression to genome stability. From deuterostomes, H2A.Z evolved into two paralogues, H2A.Z.1 and H2A.Z.2, that differ by only three amino acids and are encoded by different genes (H2AFZ and H2AFV, respectively). Despite the importance of this histone variant in development and cellular homeostasis, very little is known about the individual functions of each paralogue in mammals. Here, we have investigated the distinct roles of the two paralogues in cell cycle regulation and unveiled non-redundant functions for H2A.Z.1 and H2A.Z.2 in cell division. Our findings show that H2A.Z.1 regulates the expression of cell cycle genes such as Myc and Ki-67 and its depletion leads to a G1 arrest and cellular senescence. On the contrary, H2A.Z.2, in a transcription independent manner, is essential for centromere integrity and sister chromatid cohesion regulation, thus playing a key role in chromosome segregation.

overall_design: 3 samples were analysed: 1) Control si; 2) H2A.Z.1 si and 3) H2A.Z.2 si

first: Raquel

last: Sales-Gil

first: Dorothee

last: Kommer

first: Ines

last: de Castro

first: Hasnat

last: Amin

first: Veronica

last: Vinciotti

first: Cristina

last: Sisu


title: FACT is recruited to the +1 nucleosome of transcribed genes and spreads in a Chd1-dependent manner [ChIP-exo]

first: Francois

last: Robert

overall_design: Paired-end sequencing of exonuclease-treated chromatin immunoprecipitation (ChIP-exo) of both FACT subunits (Spt16 and Pob3), RNAPII (Rpb3) and rabbit IgG in wild type and chd1Δ cells, from two independent biological replicates (except for the IgG ChIPs which were done once), for a total of 14 ChIP-exo samples.

first: Célia

last: Jeronimo

first: Andrew

last: Angel

first: Christian

last: Poitras

first: Pierre

last: Collin

first: Jane

last: Mellor

first: François


title: Cytoplasmic NAD+ Synthesis and Ribosomal Protein Mono(ADP-Ribosyl)ation Maintain Proteostasis in Ovarian Cancer

first: W. Lee

last: Kraus

state: TX

address: 5323 Harry Hines Blvd.

institute: UT Southwestern Medical Center

summary: Global protein synthesis is upregulated in cancers, and thus cancer cells are prone to errors in translation, which may lead to protein misfolding and proteotoxic stress.  The pathways that maintain protein homeostasis support the transformation and growth of cancer cells.  Here, we show that the cytosolic NAD+ synthesis pathway (controlled by NMNAT-2) plays an essential role in ovarian cancer by maintaining protein homeostasis by regulating the activity of PARP-16, that MARylates the ribosomal proteins.  Using polysome-RNA Seq analysis, we found that inhibiting ribosomal protein MARylation by knocking down NMNAT-2 or PARP-16 leads to increased association of specific mRNAs with polysomes and enhanced translation of a set of mRNAs that contain specific secondary structures in their 3’UTRs, which leads to protein aggregation. Collectively, our study demonstrates that NMNAT-2 regulates PARP-16 activity and ribosomal protein MARylation, which control protein homeostasis by fine-tuning the levels of protein synthesis in ovarian cancers.

overall_design:
To evaluate the changes in mRNA loading on polysomes when NMNAT-2 or PARP-16 are knockdown, we performed polysome-RNA-Seq analysis on these cells. We isolated total RNA (Input RNA) and polysome-associated RNA from OVCAR3 cells subjected to NMNAT2 or PARP16 knockdown and performed RNA-Seq analysis.
Please note that each processed data was generated from both replicates and is linked to the corresponding replicate1 sample records.

first: Challa

last: Sridevi

first: Khulpateea

last: Beman

first: Nandu

last: Tulip

first: Camacho

last: Cristel V.

first: Chen

last: Hao

first: Ryu

last: Keun W.


title: FACT is recruited to the +1 nucleosome of transcribed genes and spreads in a Chd1-dependent manner [ChIP-chip]

overall_design: To investigate how FACT is recruited to chromatin in yeast Saccharomyces cerevisiae, we performed chromatin immunoprecipitation (ChIP), followed by high-resolution tiling arrays (ChIP-chip), of FACT subunits (Spt16 and Pob3) and RNAPII (Rpb3) in wild type cells. FACT and RNAPII occupancies were also measured in a temperature-sensitive mutant for RNAPII (rpb1-1) allowing to shutting down transcription after heat-shocking cells for 80 min at 37°C. To determine the contribution of CTD phosphorylation to FACT recruitment, FACT and RNAPII ChIP-chip experiments were performed in various CTD kinase mutants including kin28as, ctk1Δ, cdk8Δ, bur2Δ and the double kin28as/bur2Δ. We also profiled FACT and RNAPII using RNAPII CTD mutants where all serines at positions 2, 5 or 7 were replaced with alanines. In addition, FACT and RNAPII occupancies were measured in PAF1C mutants (paf1Δ, cdc73Δ, rtf1Δ, ctr9Δ, and leo1Δ), in cells where the N-terminal domain of Cet1 is truncated (cet1ΔN), in a temperature-sensitive mutant for the histone chaperone Spt6 (spt6-1004) and in ATP-dependent chromatin remodeler mutants (chd1Δ, isw1Δ, isw2Δ). In order to test if transcription generates a specific histone modification pattern that may be recognized by FACT, we performed FACT and RNAPII ChIP-chip experiments in sas3Δ (the catalytic subunit of NuA3), gcn5Δ, set2Δ, bre1Δ (the E3 ligase for H2B ubiquitylation), ubp8Δ (the main deubiquitylase for H2B) and ubp10Δ (a FACT-dependent H2B deubiquitylase) cells. Finally, FACT, RNAPII and Chd1 (3HA–tagged) occupancies were determined in a Chd1 catalytic dead mutant (chd1-K407R). All ChIP samples were labeled with Cy5 and hybridized against Input DNA or NoTag ChIP samples (labeled with Cy3). The microarrays used were described before (Jeronimo and Robert, 2014). The ChIP-chip data were normalized using the Limma Loess method, and replicates were combined as described previously (Ren et al. 2000, Science 290(5500):2306-9). The data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100 bp to generate data points in 10-bp intervals (Guillemette et al. 2005, PLoS Biol 3(12):e384). Each experiment was done at least from two independent biological replicates.


title: Ablation of Histone Demethylase KDM5B in Melanoma Augments Anti-Tumor Immunity through Upregulation of Retroelements

first: Qin

last: Yan


title: Ablation of Histone Demethylase KDM5B in Melanoma Augments Anti-Tumor Immunity through Upregulation of Retroelements [ChIP-seq]

summary:
Purpose: Advanced melanoma patients have poor prognosis. Although immune checkpoint blockade has revolutionized treatment for melanoma patients, majority of patients do not respond. The goal of this research is to evaluate whether epigenetic therapy targeting KDM5B could overcome resistance to immunotherapy.
Methods:ChIP-Seq analysis of KDM5B binding on retroelements in mouse melanoma cell line YUMMER1.7.
Results: We identified that KDM5B directly bind to retreoelements to repress their expression.
Conclusions: Our work characterized ablation of histone demethylase KDM5B in melanoma augments anti-tumor immunity through Upregulation of retroelements.

overall_design: ChIP-seq analysis in mouse melanoma cell lines

first: Shang-Min

last: Zhang

first: Wesley

last: Cai


title: Copper oxide nanomaterial regulation and fate in Cucurbita pepo

city: Parma

first: Luca

last: Pagano

address: Parco Area delle Scienze 11/A

department: Chemistry Life Sciences and Environmental Sustainability

country: Italy

institute: University of Parma

summary: Nanomaterials (NMs) are on the nanoscale level range ca. 1-100 nm and due to the peculiar physico-chemical properties are widely used in different areas. The present study aimed to characterize the responses of zucchini (Cucurbita pepo L.) treated with copper oxide (CuO) NPs from seed germination to the flowering stage. CuO NPs treatment did not negatively impact plant morphology and growth as well as the pollen formation and viability. Physiological analyses were followed by a complete RNAseq-based transcriptomic analysis in the different plant tissues. Mitochondrial and chloroplast functions emerged as a critical component in plant response. Evidence of CuO NPs internalization and biotransformation was a driving force to measure the impacts at both physiological and molecular levels.

overall_design: Cucurbita pepo L. (cv. Costata Romanesco) seeds were pre-germinated in vermiculite for 10 days prior to transplanting to soil. Zucchini seeds were purchased from Johnny's Selected Seeds (Albion, ME). The experimental soil was collected from the Connecticut Agricultural Experiment Station (CAES) in New Haven, CT, USA. Individual solutions of CuO NPs and CuO bulk material in water (30% water capacity of soil/vermiculite mixture) were probe sonicated to maximize dispersion. CuO bulk material and CuSO4·5H2O were purchased from Sigma Aldrich (St. Louis, MO, US). CuO NPs, CuO (bulk) or (copper sulfate) CuSO4 stock dispersions were slowly added to 500g of soil. The final concentration of CuO NPs and bulk in pots was 100 mg kg-1 while for CuSO4·5H2O (copper sulfate pentahydrate) was 320 mg kg-1, representing a total concentration of Cu about 80 mg kg-1 for all the treatments. Zucchini seedlings were planted (one each pot) and grown indoor under supplemental fluorescent lighting (60 μE m2 sec) under a photoperiod of 16h light, at approximately 22−28 °C until complete flowering. The plants were top watered for 60d growth period. Total RNA was extracted from 0.1 g of fresh plant material using a Sigma-Aldrich Spectrum Plant Total RNA Kit (Sigma-Aldrich, St. Louis, MO). Three biological replicates per treatment were extracted. After quality control samples were sent to IGA Technologies Srl (Udine, IT) for RNA sequencing service. TruSeq Stranded mRNA kit (Illumina, San Diego, CA) has been used for library preparation following the manufacturer’s instructions. RNA samples were quantified and quality tested by Agilent 2100 Bioanalyzer RNA assay (Agilent Technologies, Santa Clara, CA). Final libraries were checked Agilent Bioanalyzer DNA assay (Agilent Technologies, Santa Clara, CA). Libraries were prepared for sequencing and sequenced on single-end 75 bp mode on NextSeq 500 (Illumina, San Diego, CA). Alignment of reads to the reference transcriptome available on Cucurbitgenomics database (http://cucurbitgenomics.org/) has been performed using STAR software with default parameters. The resulting raw data have been normalized and the differentially expressed genes were identified using a 2.3 threshold of FPKM data (in log2).

first: Riccardo

last: Rossi

first: Marta

last: Marmiroli

first: Nelson


title: scRNA seq analysis of adult CD34+ cells ex vivo cultured with Delta1ext-IgG under normoxia or hypoxia

first: Daisuke

last: Araki

state: MD

address: 9000 Rockville Pike

institute: National Institutes of Health

summary:
We demonstrate that ex vivo culture of human adult hematopoietic stem and progenitor cells with Delta1ext-IgG under hypoxia limits endoplasmic reticulum stress in long-term repopulating hematopoietic stem cells and, to a lesser extent, in lineage committed progenitors compared to normoxic cultures.

Data were submitted on behalf of Dr. Larochelle's lab at the NIH.

overall_design: RNA-seq of G-CSF mobilized CD34+ cells ex vivo cultured with Delta1ext-IgG under normoxia or hypoxia

first: Andre

last: Larochelle


title: New-Onset IgG Autoantibodies in Hospitalized Patients with COVID-19 [viral]

first: Allan

last: Feng

first: Sarah

last: Chang

first: Wenzhao

last: Meng

first: Sokratis

last: Apostolidis

first: Chrysanthi

last: Skevaki

first: Eline

last: Luning Prak

first: Paul

last: Utz


title: HDAC5 loss impairs RB repression of oncogenic related genes and confers CDK4/6 inhibitor resistance in cancer

city: Rochester

first: Ting

last: Wei

state: MN

address: 507 3rd St SW

institute: Mayo Clinic


title: Genome-wide DNA demethylation of TGW (neuroblastoma cell line) after treatment of mock and 5-aza-2'-deoxycytidine (5-aza-dC)

status: Public on Jul 23 2021

first: Naoko

last: Hattori

address: 5-1-1 Tsukiji, Chuo-ku

department: Research Institute

institute: National Cancer Center

type: Methylation profiling by genome tiling array

summary: A neuroblasoma cell line, TGW, was treated with a DNA demethylating agents (5-aza-2'-deoxycytidine: 5-aza-dC). Genome-wide DNA methylation was analyzed using the Infinium Human MethylationEPIC BeadChip.

overall_design: Bisulfite converted DNA from the 2 samples were hybridized to the Infinium Human MethylationEPIC BeadChip.

first: Toshikazu

last: Ushijima


title: Ablation of Histone Demethylase KDM5B in Melanoma Augments Anti-Tumor Immunity through Upregulation of Retroelements [RNA-Seq 1445-Kdm5b KO]

first: Qin

last: Yan

first: Shang-Min

last: Zhang

first: Wesley


title: The CD155/TIGIT axis promotes and maintains immune evasion in neoantigen-expressing pancreatic cancer

city: Cambridge

first: Tyler

last: Jacks

state: MA

address: 500 Main Street

institute: MIT

summary: The CD155/TIGIT axis can be co-opted during immune evasion in chronic viral infections and cancer. Pancreatic adenocarcinoma (PDAC) is a highly lethal malignancy, and immune-based strategies to combat this disease have been largely unsuccessful to date. We corroborate prior reports that a substantial portion of PDAC harbors predicted high affinity MHC class I-restricted neoepitopes and extend these findings to advanced/metastatic disease. Using two novel preclinical models of neoantigen-expressing PDAC, we demonstrate that intratumoral neoantigen-specific CD8+ T cells adopt multiple states of dysfunction, which are similar to tumor-infiltrating lymphocytes of human PDAC patients. Mechanistically, genetic and/or pharmacologic modulation of the CD155/TIGIT axis was sufficient to promote immune evasion in autochthonous neoantigen-expressing PDAC. Finally, we demonstrate that the CD155/TIGIT axis is critical to maintain immune evasion in PDAC and uncover a combination immunotherapy (TIGIT/PD-1 co-blockade plus CD40 agonism) that elicits profound anti-tumor responses in preclinical models, now poised for clinical evaluation.

overall_design: Live CD44hiSIINFEKL_Tetramer+ CD8+ TILs from immune-evasive murine tumors were FACS sorted and subjected to scRNA-seq

first: William

last: Freed-Pastor

first: Laurens

last: Lambert

first: Zackery

last: Ely

first: Nimisha

last: Pattada

first: Arjun

last: Bhutkar

first: George

last: Eng

first: Kim

last: Mercer

first: Ana

middle: P

last: Garcia

first: Lin

last: Lin

middle: M

last: Rideout III

last: Hwang

first: Jason

last: Schenkel

first: Roderick

last: Bronson

first: Peter

last: Westcott

first: Vikram

last: Deshpande

first: Toni

last: Delorey

first: Devan

last: Phillips

first: Omer

middle: H

last: Yilmaz

first: Aviv

last: Regev


title: bulkRNA seq analysis of adult CD34+ cells ex vivo cultured with Delta1ext-IgG under normoxia or hypoxia

first: Daisuke

last: Araki

summary:
We demonstrate that a distinct hematopoietic stem cell gene expression signature was upregulated in human CD34+ cells cultured with Delta1ext-IgG in hypoxia compared to normoxic cultures.

Data were submitted on behalf of Dr. Larochelle's lab at the NIH.

first: Andre

last: Larochelle


title: Raw RNA-seq data from control and Ki-67-depleted HCT116 human colon cancer cells

city: Worcester

first: Paul

middle: D.

last: Kaufman

address: 364 Plantation St.

department: Molecular, Cell and Cancer Biology

institute: University of Massachusetts Medical School

overall_design: Cells were untreated or treated with 0.5 mM indole acetic acid (IAA) to induce degradation of the degron-tagged Ki-67 protein. Total RNA was isolated, mRNA was purified on oligo-dT beads and used to generate libraries sequenced via a PE150 protocol.

first: Osama

last: Garwain

first: Rui

last: Li

first: Lihua

last: Zhu


title: Microarray analysis of isoginkgetin-treated HCT116 and HCT116p53-/- cells

city: Ottawa

first: Bruce

last: McKay

state: Ontario

address: 1125 Colonel By Dr, Ottawa

institute: Carleton University

summary: Isoginkgetin (IGG) is a small molecule inhibitor of the spliceosome although the direct target remains elusive. Widespread failure to accurately remove introns poses a risk to cells and organisms through the potential production of aberrant mRNAs and proteins. The cellular responses to accumulation of these intermediates and/or the direct interference of spliceosome assembly itself may constitute a splicing stress but this is not well defined yet. We used oligonucleotide microarrays to assess genome wide changes in gene expression associated with exposure to IGG in HCT116 cells and an isogenic subline lacking the p53 tumor suppressor that responds to a variety of transcriptional stresses (Oncogene 18 (3), 583-592). Two of the 3 enriched pathways identified using PANTHER analysis of differentially expressed transcripts are linked to the ATF4 transcription factor and these effects are p53-independent.

overall_design: Total RNA was collected from mock-treated, vehicle control-treated (DMSO) and drug-treated (30μM IGG) HCT116 cells and a p53 null subline, following an 8 hour continuous exposure. Two independent biological experiments were performed with each cell line. An Agilent Bioanalyzer was used to ensure RNA integrity before labeling and hybridizing to Affymetrix Human Transcriptome 2.0 oligonucleotide microarrays at the Affymetrix Microarray Facility, Stemcore Laboratory, Ottawa Hospital Research Institute (Ottawa, ON, Canada). Affymetrix Transcriptome Analysis Console (TAC) 4.0 was used with default settings. A one-way between-subject unpaired ANOVA was used to determine statistical significance and was subject to false discovery rate (FDR) multi-test correction (Benjamini-Hochberg Step-Up FDR).

first: Erin

last: van Zyl

first: Victoria

last: Tolls


title: Genome-wide gene expression of NB-1 after treatment of 5-aza-dC, TBT, and combination

first: Naoko

last: Hattori

summary: A neuroblastoma cell line, NB-1, was treated with mock, a DNA demethylating agent (5-aza-2'-deoxycytidine: 5-aza-dC), a synthetic retinoic acid (tamibarotene: TBT), and the combination of 5-aza-dC and TBT. A genome-wide gene expression analysis was performed using SurePrint G3 Human Gene Expression 8 x 60K v2 Microarray.

overall_design: Cy3-labeled cRNA was synthesized from 200 ng of total RNA using a Low Input Quick Amp Labeling Kit, and 600 ng of labeled cRNA was fragmented and hybridized to SurePrint G3 Human GE 8x60K Microarray Ver2.0 Microarray.

first: Toshikazu

last: Ushijima


title: Targeting the Atf7ip-Setdb1 complex augments anti-tumor immunity by boosting tumor immunogenicity

city: New York City

first: Alireza

last: Khodadadi-Jamayran

state: NY

address: 550 1st Ave, MSB 304

department: Division of Advanced Research Technologies (DART)

institute: New York University, NYU Langone Medical Center

summary: Despite substantial progress that has been made in understanding how tumors escape immune surveillance, measures to counteract tumor immune evasion have been limited. Suppression of tumor antigen expression is a common adaptive mechanism that cancers use to evade detection and destruction by the immune system. Epigenetic modifications play a critical role in in various aspects of immune invasion, including the regulation of tumor antigen expression. To identify novel epigenetic regulators of tumor antigen expression, we established a transplantable syngeneic tumor model of immune escape with silenced antigen expression and used this system as a platform for a CRISPR-Cas9 suppressor screen targeting genes encoding epigenetic modifiers. We found that genetic disruption of the chromatin modifiers Atf7ip or its interacting partner Setdb1 in tumor cells restored tumor antigen expression, resulting in augmented tumor immunogenicity concomitant with elevated endogenous retroviral antigens (ERVs), mRNA intron retention. ERVs disinhibition was associated with a robust type I interferon response and increased T cell infiltration, leading to rejection of cells lacking intact Atf7ip or Setdb1. ATF7IP or SETDB1 expression inversely correlated with antigen processing and presentation pathways, interferon signaling, and T cell infiltration and cytotoxicity in human cancers. Our results provide a rationale for targeting Atf7ip or Setdb1 in cancer immunotherapy.

overall_design: one set of H3K9 me3 ChIP sequencing data, 7 groups with replicates including input, control, Atf7ip KO_1 Atf7ip KO_2, Setdb1 KO_2, Setdb1 KO_4 and Setdb1 KO_5  ; two set of RNA sequencing data including control and Atf7ip ko and control with Setdb1 KO (replicates for each group); one set of CRISPR loss of function screen sequencing data, replicates for each group; one set of Hi-C sequencing data including control, Atf7ip KO and Setdb1 KO

first: Hai

last: Hu

first: Fei

first: Kwok

last: Wong


title: New-Onset IgG Autoantibodies in Hospitalized Patients with COVID-19 [Unpaired Cytokine and Chemokine]

first: Allan

last: Feng

overall_design: Protein arrays were constructed by coupling antigens to carboxylated magnetic beads. Arrays consisting of conjugated beads were then used to probe hospitalized COVID-19 patient serum samples, healthy control samples, and autoimmune prototype samples  to identify autoantibodies. Samples were run in duplicate except one sample (UP67 D7) which was run in quadruplicate. for A Luminex FLEXMAP 3D® system was used to quantitatively measure autoantibody reactivity in each sample in units of mean flourescent intensity (MFI). Positive antibody hits were statistically identified by a 5 standard deviation cutoff calculated from the MFI of healthy controls.

first: Sarah

last: Chang

first: Wenzhao

last: Meng

first: Sokratis

last: Apostolidis

first: Chrysanthi

last: Skevaki

first: Eline

last: Luning Prak

last: Utz


title: The gut microbiota instructs the hepatic endothelial cell transcriptome

city: Kiel

first: Joana

middle: Pimenta

last: Bernardes

address: Am Botanischen Garten 11

department: Systems Immunology

country: Germany

institute: Institute of Clinical Molecular Biology

summary: The gut microbiota affects remote organ functions but its impact on organotypic endothelial cell (EC) transcriptomes remains unexplored. The liver endothelium encounters microbiota-derived signals and metabolites via the portal circulation. To pinpoint how gut commensals affect the hepatic sinusoidal endothelium, a magnetic cell sorting protocol, combined with fluorescence activated cell sorting, was used to analyze the transcriptome of hepatic sinusoidal ECs from germ-free (GF) and conventionally-raised (CONV-R) mice by RNA-sequencing. This resulted in a comprehensive map of microbiota-regulated hepatic EC-specific transcriptome profiles. Gene Ontology analysis revealed that several functional processes in the hepatic endothelium were influenced. The absence of a microbiota influenced the expression of genes involved in cholesterol flux and angiogenesis. Specifically, genes functioning in hepatic endothelial sphingosine matabolism and the sphingosine-1-phosphate pathway showed a drastically increased expression in the GF state. Our analyses reveal a prominent role for the microbiota in shaping the transcriptional landscape of the hepatic endothelium.

overall_design: Transcriptome  analysis of hepatic sinusoidal ECs from germ-free (GF) and conventionally-raised (CONV-R) mice by RNA-sequencing

first: Henning

last: Formes

first: Amrit

last: Mann

first: Franziska

last: Bayer

first: Giulia

last: Pontarollo

first: Klytaimnistra

last: Kiouptsi

first: Katrin

last: Schäfer

first: Sebastian

last: Attig

first: Teodora

last: Nikolova

first: Thomas

middle: G

last: Hofmann

first: Jörn

last: Schattenberg

first: Hristo

last: Todorov

first: Susanne

last: Gerber

first: Philip

last: Rosenstiel

first: Tobias

last: Bopp

first: Felix

last: Sommer

first: Christoph

last: Reinhardt


title: GAR-Driven Subcellular Localization of the Growth Controlling RNA Binding Protein Nucleolin

city: Los Angeles

first: Riki

last: Kawaguchi

address: 760 Westwood Plaza, Room 37-420

department: Department of Neurology and Department of Psychiatry

institute: University of California Los Angeles

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: HDAC5 loss impairs RB repression of oncogenic related genes and confers CDK4/6 inhibitor resistance in cancer [ChIP-seq]

first: Ting

last: Wei

summary: The tumor suppressor protein RB acts as a transcription repressor via the interaction of its pocket domain with an L/IXCXE motif in HDAC proteins such as HDAC1. Here we demonstrated that while lacking an L/IXCXE motif, HDAC5 interacts with both RB-N (via an FXXXV motif) and RB-C segments and this interaction is diminished by RB serine-249/threonine-252 and threonine-821 phosphorylation. HDAC5 gene is frequently downregulated or deleted in human cancers such as prostate cancer. Loss of HDAC5 increases histone H3 lysine 27 acetylation (H3K27-ac) and circumvents RB-mediated repression of cell cycle-related oncogenic genes. Accordingly, HDAC5 loss confers resistance to the CDK4/6 inhibitors such as Palbociclib in prostate cancer cells in vitro and in mice, but this effect is overcome by the BET-CBP/p300 dual inhibitor NEO2734. Our findings reveal an unknown role of HDAC5 in RB-mediated histone deacetylation and gene repression and a mechanism modulating CDK4/6 inhibitor therapeutic sensitivity in cancer cells.

overall_design: PC-3 cells treated with DMSO or Palbociclib for 24h were harvested for ChIP-seq.

first: Haojie

last: Huang

first: Yingke

last: Zhou

first: Donglin

last: Ding

first: Liguo

last: Wang


title: Mutant collagen COL11A1 enhances cancerous invasion

first: Paul

last: Khavari

state: California

address: 269 Campus Drive CCSR 2150

department: Dermatology

institute: Stanford University School of Medicine

type: Genome variation profiling by high throughput sequencing


title: Analysis of RNA localization in nucleolin deficient axons

summary: We previously showed that the RNA binding protein Nucleolin is localized to axons of DRG sensory neurons by interaction with the molecular motor Kinesin-1 (Kif5A) and subsequently localizes importin beta1 mRNA there (Perry et al., 2016). In this work, we created a mouse with a heterozygous deletion of the domain in nucleolin responsible for the Kif5A interaction and showed that nucelolin protein levels are reduced in the axons of dorsal root ganglia neurons in this mouse model. The goal of this study was to identify RNAs who's axonal localization is affected by this perturbation.

overall_design: Total RNA from the neurite or soma compartments of dorsal root ganalia neurons cultured on modified Boyden chambers was collected and analysed by RNA sequencing. We repeated this in 3 independent biological repeats.

first: Ella

last: Doron-Mandel

first: Ofri

last: Abraham

first: Indrek

last: Koppel

first: Giovanni

last: Coppola

first: Michael

last: Fainzilber


title: Biallelic RNU12 noncoding variants cause CDAGS syndrome

first: Donald

last: Glass

address: 5323 Harry Hines Blvd, Mail Code 9069

summary: CDAGS Syndrome is a rare congenital disorder characterized by Craniosynostosis, Delayed closure of the fontanelles, cranial defects, clavicular hypoplasia, Anal and Genitourinary malformations, and Skin manifestations. We performed exome sequencing to identify the underlying molecular cause in five patients with CDAGS syndrome from four distinct families. Whole exome sequencing revealed rare variants that disrupt highly conserved nucleotides within the RNU12 gene. RNU12 encodes a small nuclear RNA that is a component of the minor spliceosome and is essential for minor intron splicing. Targeted sequencing confirmed allele segregation within the four families. All five patients in this cohort have a rare variant on one allele that either disrupts the secondary structure or the Sm binding site of the RNU12 snRNA. The variant on the other allele, shared among all five cases, alters a highly conserved nucleotide within the precursor U12 snRNA 3’ extension that is absent in 1440 unrelated healthy controls. All of the variants are either rare or absent from all searched public databases. Whole transcriptome sequencing analysis identified gene dysregulation and specific defects in intron retention in a subset of minor intron splicing. These findings provide evidence of the involvement of RNU12 in craniosynostosis, anal and genitourinary patterning and cutaneous disease.

overall_design: RNA profile of lymphoblastoid cell lines from CDAGS proband, two unaffected family members, and two unrelated unaffectd controls -- generated by deep sequencing

first: Chao

last: Xing

first: Mohammed

last: Kanchwala


title: Analysis of Nucleolin and PTBP1 RNA targets before after Sciatic nerve injury

summary: We previously showed that importin beta1 local translation in injured axons is a key event in injury-signaling and regeneration (Perry et al., 2012). Next, we showed that importin beta1 is localize to axons through a localization motif in its 3'UTR and that this motif is bound by the RNA binding proteins Nucleolin and PTBP1. In this study, we asked which other RNA targets are bound by these two RNA binding proteins in naive Sciatic nerves and how are they changing 7 days after injury.

overall_design: We immunoprecipitated Nucleolin and PTBP1 from naive or injured Sciatic nerves (7 days after injury), isolated the bound RNA and analyzed it by RNA sequencing. As controls, we pre-incubated the antibodies used for immunoprecipitation with their respective blocking peptides, so as to block their ability to bind the target proteins. We also sequenced as reference a fraction of the input material from each sample. We pooled 10 Sciatic nerves per sample and repeated this experiment in 3 independent biological repeats.

first: Stefanie

last: Alber


title: HDAC5 loss impairs RB repression of oncogenic related genes and confers CDK4/6 inhibitor resistance in cancer [RNA-seq]

overall_design: PC-3 cells transfected with shNC or shHDAC5 were treated with DMSO or Palbociclib for 24h and were harvested for RNA-seq.


title: COL11A1 mutations in cutaneous squamous cell carcinoma [WES]

summary: Whole exome sequencing was used to identify frequent COL11A1 mutations in cutaneous squamous cell carcinoma (cSCC).

overall_design: Genomic DNA was then isolated from human cSCC using the DNeasy Blood and Tissue kit (Qiagen) according to the manufacturer’s instructions. Sequencing libraries were prepared using the Agilent SureSelect Human All Exon V5 (51Mb) target enrichment for whole exome capture and sequenced on the Illumina HiSeq platform with 2x100nt paired-end reads.

first: Carolyn

last: Lee

first: Tomas

last: Bencomo

first: Zurab

last: Siprashvili


title: Profiling Kif5A RNA targets in sensory neuron axons

first: Riki

last: Kawaguchi

summary: We previously identified that the RNA binding protein Nucleolin is localized to axons of DRG sensory neurons by interaction with the molecular motor Kinesin-1 (Kif5A) and subsequently localizes importin beta1 mRNA there (Perry et al., 2016). To further identify additional RNAs that are localized to axons in a similar mechanism, we immunopercipitated Kif5A from dorsal roots (centrally projecting axons) or Sciatic nerves (peripherally projecting axons), isolated the bound RNA and sequnced it.

overall_design: We conducted Kif5A immunopercipitation from axoplasm using a rabbit polyclonal antibody. We used a rabbit isotype control (IgG) and also sequenced some of the input material. Axoplasm was extracted either from Dorsal roots (DR) or Sciatic nerves (SN) of the same animal, and 5 animals were pulled per biological repeat. We repeated this in 3 independent biological repeats.


title: Marginal zone B cells leverage the Notch2-Myc-mTORC1 signaling axis to achieve division independent plasma cell differentiation.

status: Public on Jul 22 2021

city: Philadelphia

first: Brian

middle: Thomas

last: Gaudette

state: PA

address: 3620 Hamilton Walk, Pathology and Laboratory Medicine

department: Pathology and Lab Medicine

institute: University of Pennsylvania


title: Single-cell  transcriptomic profiles of CD45-positive cells from Mx1-Cre x Adam10f/f Rag2KO mice

first: Keisuke

last: Nagao

summary: Induced ablation of Adam10 in Mx1-Cre x Adam10f/f mice results in T cell-independent Inflammatory alopecia.  Mx1-Cre x Adam10f/f mice were crossed to the Rag2KO background. After poly(I:C) injection, mouse skin was dissociated and sorted for CD45-positive cells which were subjected to single-cell RNA sequencing analysis.

overall_design: Single-cell transcriptomic profiles of CD45-positive cells sorted from Mx1-Cre x Adam10f/f Rag2KO mouse skin 30 days after poly(I:C) injection.

first: Keiko

last: Sakamoto

first: Seon-Pil

last: Jin


title: Astrocyte-derived TNF and glutamate critically modulate microglia activation by methamphetamine

city: Porto

first: Teresa

last: Canedo

address: R. Alfredo Allen 208

country: Portugal

summary: Methamphetamine (Meth) is a powerful illicit psychostimulant, widely used for recreational purposes. Besides disrupting the monoaminergic system and promoting oxidative brain damage, Meth also causes neuroinflammation, contributing to synaptic dysfunction and behavioral deficits. Aberrant activation of microglia, the largest myeloid cell population in the brain, is a common feature in neurological disorders triggered by neuroinflammation. In this study, we investigated the mechanisms underlying the aberrant activation of microglia elicited by Meth in the adult mouse brain. We found that binge Meth exposure caused microgliosis and disrupted risk assessment behavior (a feature that usually occurs in individuals who abuse Meth), both of which required astrocyte-to-microglia crosstalk. Mechanistically, Meth triggered a detrimental increase of glutamate exocytosis from astrocytes (in a process dependent on TNF production and calcium mobilization), promoting microglial expansion and reactivity. Ablating TNF production, or suppressing astrocytic calcium mobilization, prevented microglia reactivity and abolished the behavioral phenotype elicited by Meth in the elevated plus maze (EPM). Overall, our data indicate that glial crosstalk is critical to relay alterations caused by acute Meth exposure.

overall_design: Microglia (Cd11b+, CD45low, and CD206-) from WT Saline (3 mice) or WT treated with Meth (3mice) were FACS-isolated and total RNA was collected. Sequencing was performed on an Ion S5XL™ sequencer (Thermo Scientific).

first: Camila

middle: C

last: Portugal

first: Renato

last: Socodato

first: Tiago

middle: O

last: Almeida

first: Ana

middle: F

last: Terceiro

first: Joana

last: Bravo

middle: I

last: Silva

first: João

last: Magalhães

first: Sónia

last: Guerra-Gomes

last: Oliveira

first: Nuno

last: Sousa

last: Relvas

last: Summavielle


title: Alterations in circulating monocytes predict COVID-19 severity and include chromatin modifications still detectable six months after recovery

city: Madrid

first: Alberto

last: Utrero-Rico

address: Av Cordoba s/n

country: Spain

summary: An early analysis of circulating monocytes may be critical for predicting COVID-19 course and its sequelae. In 131 untreated, acute COVID-19 patients at emergency room (ER) arrival, monocytes showed decreased surface molecules expression, including HLA-DR, in association to an inflammatory cytokine status and limited anti-SARS-CoV-2-specific T cell response. These alterations were mostly normalized in post-COVID-19 patients, 6 months after discharge. Acute COVID-19 monocytes transcriptome showed upregulation of anti-inflammatory, tissue repair genes such as BCL6, AREG and IL-10 and increased accessibility of chromatin. Some of these transcriptomic and epigenetic features still remained in post-COVID-19 monocytes. Importantly, a poorer expression of surface markers and low IRF1 gene transcription in circulating monocytes at ER defined a COVID-19 patients group with impaired SARS-CoV-2-specific T cell response and increased risk of requiring intensive care or dying.  An early analysis of monocytes may be useful for COVID-19 patients stratification and to designing innate immunity-focused therapies.

overall_design: We performed Nanostring analysis using fresh isolated monocytes from Control patients with no Covid-19 infection, Acute Covid-19 or Post Covid-19 infection

first: Cecilia

last: Gonzalez-Cuadrado

first: Marta

last: Chivite-Lacaba

first: Oscar

last: Cabrera-Marante

first: Rocio

last: Laguna-Goya

first: Patricia

last: Almendro-Vázquez

first: Carmen

last: Díaz-Pedroche

first: Maria

last: Ruiz-Ruigomez

first: Antonio

last: Lalueza

first: María

last: Dolores Folgueira

first: Enrique

last: Vázquez

last: Quintas

first: Marcos

last: Berges-Buxeda

first: Moisés

last: Martín-Rodríguez

last: Dopazo

last: Serrano-Hernández

first: José

last: Aguado

first: Estela

last: Paz-Artal


title: The Pol II Pre-Initiation Complex (PIC) Influences Mediator Binding but Not Promoter-Enhancer Looping [nascent RNA-seq]

first: Fei

last: Sun

address: 615 Charles E Young Drive South

department: Biological Chemistry

institute: UCLA

summary: Knowledge of how Mediator and TFIID crosstalk contributes to promoter-enhancer (P-E) communication is important for elucidating the mechanism of enhancer function. We conducted an shRNA knockdown screen in murine embryonic stem cells to identify the functional overlap between Mediator and TFIID subunits on gene expression. Auxin-inducible degrons were constructed for TAF12 and MED4, the subunits eliciting the greatest overlap. Degradation of TAF12 led to a dramatic genomewide decrease in gene expression accompanied by destruction of TFIID, loss of Pol II pre-initiation complex (PIC) at the promoter, and significantly decreased Mediator binding to promoters and enhancers. Interestingly, loss of the PIC elicited only a mild effect on P-E looping by Promoter Capture Hi-C (PCHi-C).  Degradation of MED4 had a minor effect of Mediator integrity but led to a consistent 2-fold loss in gene expression, decreased binding of Pol II to Mediator, and decreased recruitment of Pol II to the promoters, but no effect on the other PIC components. PCHi-C revealed no consistent effect of Med4 degradation on P-E looping. Collectively, our data show that TAF12 and MED4 contribute mechanistically in different ways to P-E communication but neither factor appears to directly control P-E looping, thereby dissociating P-E communication from physical looping.

overall_design: Each subunit of TFIID and Mediator was knocked down individually using shRNA in V6.5 cells. mRNA-seq was employed to identify key subunits that contribute more to global transcription and elicit the greatest overlap. Degron cell lines were constructed for the identified key subunits TAF12 and MED4. Nascent RNA-seq, ChIP-seq and promoter capture Hi-C were performed using these cells either treated or untreated by auxin.

first: Michael

last: Carey


title: The Pol II Pre-Initiation Complex (PIC) Influences Mediator Binding but Not Promoter-Enhancer Looping [promoter_capture_Hi-C]


title: Transcriptomic analyis of in-vitro plasma cell generation from follicular and marginal zone B cells with cell cycle inhibition via PD0332991 and rapamycin

first: Brian

last: Gaudette

summary: Plasma cell differentiation is thought to be tied to cell division. We observe that marginal zone B cells are able to differentiate to plasma cells without completing a single cell division.  In order to characterize the transcriptome of plasma cells derrived from un-divided MZ B cells we performed in vitro plasma cell differentiation cultures in the presence of the CDK4/6 inhibitor PD0332991 or rapamycin using Blimp1-GFP reporter follicular and MZ B cells. We report here that these resulting plasma cells from undivided MZ B cells exhibit normal plasma cell gene expression and function in the absence of mitosis.

overall_design: Samples are follicular B cells and marginal zone B cells stimulated in culture to produce plasma cells.  cDNA was prepared from RNA with 3' polyA capture method.  Transcript abundance was measured by Kallisto psuedo-alignment and normalized (voom) and differential expression was assessed using limma.

first: David

last: Allman


title: Transcriptomic analyis of follicular and marginal zone B cells after inducible Tsc1 deletion

summary: Observations that ongoing mTORC1 activity drives plasma cell poise in MZ B cells we rationed that enforcing mTORC1 activity on mature follicular B cells by inducible B cell specific Tsc1 deletion would confer plasma cell differention poise on follicular B cells.  We therefore performed RNA-seq on follicular and marginal zone B cells from Tsc1F.hCD20-TamCre mice after 4 weeks tamoxifen treatment to examine transcriptional markers of plasma cell differentiation poise.

overall_design: Samples are sorted follicular and marginal zone B cells from Tsc1-deleted, wild type and hets after 4 weeks induction by tamoxifen. cDNA was prepared from RNA with 3' polyA capture method.  Transcript abundance was measured by Kallisto psuedo-alignment and normalized (voom) and differential expression was assessed using limma.


title: The Pol II Pre-Initiation Complex (PIC) Influences Mediator Binding but Not Promoter-Enhancer Looping [RNA-seq]


title: The Pol II Pre-Initiation Complex (PIC) Influences Mediator Binding but Not Promoter-Enhancer Looping [ChIP-seq]


title: A Gata3 enhancer necessary for ILC2 development and function

city: Chicago

first: Albert

last: Bendelac

state: IL

address: 924 E. 57th Street BSLC

institute: University of Chicago

summary: The type 2 helper effector program is driven by the master transcription factor GATA3 and can be expressed by subsets of both innate lymphoid cells (ILCs) and adaptive CD4+ T helper (Th) cells. While ILC2s and Th2 cells acquire their type 2 differentiation program under very different contexts, the distinct regulatory mechanisms governing this common program are only partially understood. Here we show that the differentiation of ILC2s, and their concomitant high level of GATA3 expression, are controlled by a Gata3 enhancer, Gata3 +674/762, that plays only a minimal role in Th2 cell differentiation. Mice lacking this enhancer exhibited defects in several but not all type 2 inflammatory responses, depending on the respective degree of ILC2 and Th2 cell involvement. Our study provides molecular insights into the different gene regulatory pathways leading to the acquisition of the GATA3-driven type 2 helper effector program in innate and adaptive lymphocytes.

overall_design:
ATAC-seq was performed in ILC precursors from bone marrow of Tcf7-mCherry reporter mice

Cell population definitions:
CLP - Common Lymphoid Precursor
Flt3p_aLP - Flt3 positive alpha4beta7 Lymphoid Precursor
rEILP - Refined Early Innate Lymphoid Precursor
iILCP - Incipient Innate Lymphoid Cell (ILC) Precursor
ILCP - ILC Precursor
ILC2P - ILC2 Precursor

first: Darshan

middle: N

last: Kasal


title: Mutant collagen COL11A1 enhances cancerous invasion [RNA-seq]

first: Paul

last: Khavari

summary: RNA-seq was used to define the role of mutant COL11A1 in cutaneous squamous cell carcinoma (cSCC) by comparing human cSCC organoids edited to introduce the COL11A1 G598A substitution to organoids edited with an A to T substitution that preserves the native glycine residue while controlling for non-specific editing effects.

overall_design: Total RNA was then isolated from human SCC organoids using QIAshredder (Qiagen) and Trizol (ThermoFisher) followed by DNA removal with the TURBO DNA-free kit (Ambion) according to the manufacturer’s instructions. RNA integrity was verified using an Agilent 2100 Bioanalyzer. RNA-Seq libraries were prepared with the mRNA Seq Sample Prep Kit (Illumina, Inc.) as recommended by the manufacturer. 75 bp paired-end sequencing reads were obtained using the Illumina HiSeq platform.

first: Carolyn

middle: S

last: Lee

first: Tomas

middle: J

last: Bencomo

first: Zurab

last: Siprashvili


title: Transcription-mediated supercoiling regulates genome folding and loop formation

city: Barcelona

first: Maria

middle: Pia

last: Cosma

address: Dr Aiguader 88

department: Gene Regulation, Stem Cells and Cancer

institute: Centre for Genomic Regulation


title: Transcription-mediated supercoiling regulates genome folding and loop formation [ChIP-Seq]

summary: The chromatin fiber folds into loops, however the mechanisms controlling loop extrusion are still poorly understood. Using super-resolution microscopy, we visualized that loops in intact nuclei are formed by a scaffold of cohesin complexes from which the DNA protrudes. RNA polymerase II decorates the top of the loops and is physically segregated from cohesin. Augmented looping upon increased loading of cohesin on chromosomes causes disruption of Lamin at the nuclear rim and chromatin blending, a homogenous distribution of chromatin within the nucleus. Altering supercoiling via either transcription or topoisomerase inhibition counteracts chromatin blending, increases chromatin condensation, disrupts loop formation and leads to altered cohesin distribution and mobility on chromatin. Overall, negative supercoiling generated by transcription is an important regulator of loop formation in vivo.

overall_design: ChIP profiling of H3K4me2 histone mark in HeLa cells (control DMSO treated)

first: Melike

last: Lakadamyali

middle: V

last: Neguembor


title: STAU2 Binds a Complex RNA Cargo that Changes Temporally With Production of Diverse Intermediate Progenitors During Mouse Corticogenesis

city: Rensselaer

first: Rebecca

last: Chowdhury

address: 1 Discovery Drive

institute: Neural Stem Cell Institute

summary: The double-stranded RNA binding protein (RBP) STAU2 displays temporally regulated associations with RNA determinants that contribute to IPC and cortical lamination during corticogenesis.

overall_design: Examination of RNAs bound to STAU2 across four stages (E11.5, E13.5, E15.5, E17.5) of mouse cortical development.

first: Nathan

last: Boles

first: Sally

last: Temple


title: Transcriptomic analyis follicular and marginal zone B ells after in vivo Notch2 blockade.

summary: Marginal zone B cells which make rapid mitosis-independent plasma cell responses are dependent on Notch2 signaling for their generation and maintenance.  In order to determine the effect of ongoing Notch signaling on the maintenance of the MZ transcriptome, we performed RNA-seq on MZ and follicular B cells after 12, 24 and 48 hours of in vivo Notch2 antibody blockade

overall_design: Samples are sorted marginal zone and follicular B cells from animals after 12, 24 and 48 hours in vivo anti-Notch2 antibody treatment or isotype control. cDNA was prepared from RNA with 3' polyA capture method.  Transcript abundance was measured by Kallisto psuedo-alignment and normalized (voom) and differential expression was assessed using limma.

middle: T


title: Transcription-mediated supercoiling regulates genome folding and loop formation [RNA-Seq]

overall_design: mRNA profile of HeLa cells (control DMSO treated)

middle: P


title: Codependency and mutual exclusivity for gene community detection from sparse single-cell transcriptome data

city: Minato-ku

first: Natsu

last: Nakajima

state: Tokyo

address: General Research Building 8th floor, 4-6-1, Shirokanedai

department: Institute of Medical Science

institute: The University of Tokyo

summary: Single-cell RNA-seq (scRNA-seq) can be used to characterize cellular heterogeneity in thousands of cells. The reconstruction of a gene network based on coexpression patterns is a fundamental task in scRNA-seq analyses, and the mutual exclusivity of gene expression can be critical to understand such heterogeneity. Here, we propose an approach for detecting communities from a gene network constructed on the basis of coexpression properties. The community-based comparison of multiple coexpression networks enables the identification of functionally related gene clusters that cannot be fully captured through differential gene expression-based analysis. We also developed a novel metric referred to as the exclusively expressed index (EEI) that identifies mutually exclusive gene pairs from sparse scRNA-seq data. EEI quantifies and ranks the exclusive expression levels of all gene pairs from binary expression patterns while maintaining robustness against a low sequencing depth. We applied our methods to glioblastoma scRNA-seq data and found that gene communities are partially conserved after serum stimulation despite a considerable number of differentially expressed genes. We also demonstrate that the identification of mutually exclusive gene sets with EEI can improve the sensitivity of capturing cellular heterogeneity. Our methods complement existing approaches and provide new biological insights from even a large sparse dataset in the single-cell analysis field.

overall_design: GSCs were cultured in Dulbecco’s modifiedEagle’s medium (DMEM)/F12 (Life Technologies) containing a B27 supplement minus vitamin A (Life Technologies), epidermal growth factor, and fibroblast growth factor 2 (20 ng/ml each; Wako Pure Chemicals Industries). For in vitro differentiation, GSCs were cultured in Dulbecco’s modified Eagle’s medium/F-12 medium (Life Technologies) containing 10% fetal bovine serum for the indicated times. Single-cell suspensions of GSCs or serum-induced differentiated GSCs were subjected to droplet-based scRNA-seq library preparation with the Chromium Single Cell 3' Reagent Kit v2 (10x Genomics), aiming for an estimated 2,000 cells per library and following the manufacturer’s instructions. The libraries were checked with a BioAnalyzer High Sensitivity Chip (Agilent), quantified with a KAPA Library Quantification kit (Roche), and then sequenced on the Illumina Hiseq 2500 platform in rapid mode.

first: N

first: T

last: Hayashi

first: K

last: Fujiki

last: Akiyama

first: R

last: Nakato


title: Harmful R loops are prevented via different cell cycle specific mechanisms

city: Seville

first: Andrés

last: Aguilera

address: Av. Américo Vespucio 24

department: Genome BIology

institute: CABIMER, Universidad de Sevilla

summary: R loops induce transcription-replication (T-R) conflicts, but recent reports have suggested that R-loops are caused by T-R conflicts. Identifying how R loops are generated is crucial to know how transcription compromise genome integrity. Genome-wide analyses of conditional yeast mutants reveal that transcription factors, such as the THO complex. prevents R loop formation in G1 and S-phase, whereas the Sen1 DNA-RNA helicase prevents them only in S-phase. Interestingly, damage accumulates asymmetrically downstream of the RF in sen1 cells but symmetrically in the hpr1 THO mutant. Our results unambiguously indicate that: R loops forms co-transcriptionally independently of DNA replication; THO is a general and cell-cycle independent safeguard against R loops, and Sen1, in contrast to previously believed, is an S-phase-specific R loop resolvase. These conclusions have important implications for the mechanism of R loop formation and the role of other factors reported to impact on R loop homeostasis

overall_design: DNA-RNA immunoprecipitation strand-specific (DRIPc-seq) were performed on WT YMK612 and hpr1 and sen1 depleted cells from G1-synchronized or S-phase cultures, treated or not with Rnase H. H2AP ChIP-seq were performed on WT YMK612 and hpr1 and sen1 depleted cells from S-phase cultures.

first: Marta

last: San Martin-Alonso

middle: E

last: Soler-Oliva

last: Garcia-Rubio

first: Tatiana

last: Garcia-Muse

first: Andres


title: Human monoclonal antibodies to M. tuberculosis arabinomannan recognize distinct glycan epitopes - implications for TB diagnostics and research

status: Public on Jul 21 2021

city: Bronx

first: Jacqueline

last: Achkar

address: 1300 Morris Park Ave.

institute: Albert Einstein College of Medicine

summary:
Human antibody responses to AM/LAM are heterogenous and knowledge of reactivity to specific glycan epitopes at the monoclonal level is limited.
Using novel glycan arrays, we characterized very high affinity monoclonal antibodies to AM/LAM,  determined these mAbs are non-competing, and recognized distinct glycan epitopes. distinct from other anti-AM/LAM mAbs reported.

sample_organism:
Homo sapiens
Mus musculus

overall_design: Monoclonal antibodies at 5 ug/ml were tested for direct binding to 63 synthetic oligosaccharides

first: Elise

last: Ishida

first: Maju

last: Joe

first: Todd

middle: L

last: Lowary

middle: M


title: The Pol II prenitiation complex (PIC) influences Mediator binding but not promoter-enhancer looping

first: Fei

last: Sun

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Liver-dependent lung remodeling in the wake of systemic inflammation shapes responses to secondary infection

city: Boston

first: Boston University

last: Microarray and Sequencing Resource

address: 72 East Concord Street, E631

department: Microarray and Sequencing Resource

institute: Boston University

summary: Systemic duress such as that elicited by sepsis, burns or trauma predispose patients to nosocomial pneumonia, demanding a better understanding of host pathways influencing this connection. These systemic challenges are also capable of triggering the hepatic acute phase response (APR), an event that we have previously demonstrated as essential for limiting susceptibility to secondary lung infections. In an effort to identify potential mechanisms underlying protection afforded by the lung-liver axis, our current studies aimed to comprehensively evaluate liver-dependent lung reprogramming following a systemic inflammatory challenge with endotoxemia followed by pneumonia. To do so, WT mice and APR-deficient littermates lacking hepatocyte STAT3 (hepSTAT3-/-), a transcription factor necessary for full APR initiation, were challenged intraperitoneally with LPS to induce endotoxemia. After 18h, pneumonia was induced by an intratracheal E. coli instillation. Lung transcriptional profiling, airspace proteomics, and additional validation measures were assessed after endotoxemia with or without a subsequent challenge with pneumonia in order to achieve an unbiased assessment of lung immune activity in the presence and absence of an intact liver response. In WT mice, endotoxemia elicited robust transcriptional changes in the lungs, with nearly 2,000 differentially expressed genes when comparing WT and hepSTAT3-/- mice. The resulting gene signatures revealed highly exaggerated immune activity in the lungs of hepSTAT3-/- mice, which were compromised in their capacity to launch additional cytokine responses to secondary infection.  Proteomics also revealed substantial liver-dependent modifications in the airspaces of pneumonic mice, implicating a network of dispatched liver-derived mediators influencing lung homeostasis. These results indicate that following a systemic inflammatory event, liver acute phase changes dramatically remodel the lungs, resulting in a modified landscape for any stimuli encountered thereafter. Based on the known vulnerability of hepSTAT3-/- mice to secondary lung infections, we believe that intact liver function is critical for maintaining the immunological responsiveness of the lungs. However, further studies are needed to confirm whether and how such lung changes directly influence pneumonia susceptibility.

overall_design: Transcriptional profiles of wild type (WT) and hepSTAT3-/- mice with 18h i.p. LPS alone or with 24h i.t. E. coli, and an additional control group of WT i.p. saline

first: Christine

last: Odom

first: Lee

last: Quinton


title: Neurotranscriptomic changes associated with chick-directed parental care in adult non-reproductive Japanese quail

city: Orange

first: Patricia

last: Lopes

address: 450 N Center St

department: Biological Sciences

institute: Chapman University

summary:
For many species, parental care critically affects offspring survival. But what drives animals to display parental behaviours towards young? In mammals, pregnancy‐induced physiological transformations seem key in preparing the neural circuits that lead towards attraction (and reduced‐aggression) to young. Beyond mammalian maternal behaviour, knowledge of the neural mechanisms that underlie young‐directed parental care is severely lacking. We took advantage of a domesticated bird species, the Japanese quail, for which parental behaviour towards chicks can be induced in virgin non‐ reproductive adults through a sensitization procedure, a process that is not effective in all animals. We used the variation in parental responses to study neural transcriptomic changes associated
with the sensitization procedure itself and with the outcome of the procedure (i.e., presence of parental behaviours). We found differences in gene expression in the hypothalamus and bed nucleus of the stria terminalis, but not the nucleus taeniae. Two genes identified are of particular interest. One is neurotensin, previously only demonstrated to be causally associated with maternal care in mammals. The other one is urocortin 3, causally demonstrated to affect young‐directed neglect and aggression in mammals. Because our studies were conducted in animals that were reproductively quiescent, our results reflect core neural changes that may be associated with avian young‐directed care independently of extensive hormonal stimulation. Our work opens new avenues of research into understanding the neural basis of parental care in non‐placental species.

overall_design: RNA-seq dataset from three brain regions (hypothalamus, nucleus taena and bed nucleus of the stria terminalis) from adult quail exposed (I) or not (C) to a parental sensitization treatment. Males (M) and females (F) were included in each treatment.


title: Single-cell RNA sequencing of murine Colon26 tumors after combination treatment with HMGN1 immunostimulatory peptide (minP1) with anti-PD-L1 antibody

first: Alex Chang-Yu

last: Chen

address: 149 13th Street, 7th floor, Room 7103

department: Krantz Family Center for Cancer Research

institute: Massachusetts General Hospital

summary: In this study, we explore a new combination therapy of anti-PD-L1 antibody with an immunostimulatory peptide derived from an alarmin high mobility group nucleosome binding protein 1 (HMGN1). Combination treatment of HMGN1 immunostimulatory peptide (minP1) with anti-PD-L1 antibody exerted robust anti-tumor effects in the Colon26 subcutaneous murine model. To understand transcriptomic differences of the immune cell populations in the tumor-bearing mice after treatment with single-agent or combination therapy of minP1 with anti-PD-L1 antibody, we performed single-cell RNA sequencing (scRNA-seq) analysis on CD45+ immune cell populations using the BD Rapsody system, NEBNext UltraII FS library prep kit, and a Illumina Novaseq 6000 S4 flowcell.

overall_design:
Single-cell RNA sequencing (scRNA-seq) was performed on CD45+ cells from the Colon26 tumors of BALB/c mice after treatments. There are untreated (n=3, biological replicates),  minP1-treated (n=4, biological replicates), anti-PD-L1 antibody-treated (n=3, biological replicates), and minP1/anti-PD-L1 antibody-treated (n=4, biological replicates) groups. Intratumoral CD45+ cells from different treatment groups were stained with Hashtag anti-CD45 antibody to separate each sample first,  were processed in separate experimental runs of the BD Rapsody system, were performed single-cell cDNA library by using NEBNext UltraII FS library prep kit, and were run by an Illumina Novaseq 6000 S4 flowcell (67 bp read1 and 140 bp read2) (Illumina, USA) to a depth of approximately 100,000 reads/cell. , which finally results in 14 samples across which 7,613 cells were passed through quality check and were analyzed by Seurat. For mouse data, after adapter removal by using Cutadapt-2.10 (ref), gene-expression libraries were aligned to the mm10 reference transcriptome by using Bowtie2-2.3.4.1, and count matrices were generated using home-built shell scripts and the modified python script of BD Rhapsody workflow. Valid cell barcodes were identified as cell barcodes above inflection threshold of knee-plot of total read counts of each cell barcode identified by DropletUtils package (Griffiths JA et al. Nat. Commun. 2018). Each sample origin and doublets were identified based on fold-change of the normalized read counts of Hashtag antibodies. Data were log-transformed [log(TPM + 1)] for all downstream analyses, most of which were performed using the R software package Seurat v2.3.4 (http://satijalab.org/seurat) to analyze immune cell populations.
This submission represents 14 unique/individual tumor-bearing mice

first: Chang-Yu

first: Shigeyuki

last: Shichino

first: Ueha

last: Satoshi

first: Yoshiro

last: Ishiwata

first: Shoji

last: Yokochi

first: De

last: Yang

first: Joost

last: Oppenheim

first: Haru

last: Ogiwara

first: Shungo

last: Deshimaru

first: Yuzuka

last: Kanno

first: Tatsuro

last: Ogawa

first: Shiro

last: Shibayama

first: Kouji

last: Matsushima


title: DE-ETIOLATED1 has a role in the circadian clock of the liverwort Marchantia polymorpha

city: Uppsala

first: Magnus

last: Eklund

address: Norbyv. 18D

country: Sweden

institute: Uppsala University

summary: Identification of  genes with circadian rhythm in the liverwort Marchantia polymorpha subsp. ruderalis

overall_design: Time series of mRNA profiles from thallus tissue grown in constant light conditions

first: Ulf

last: Lagercrantz

first: Anja

last: Billhart

first: Sabine

last: Rousku

first: Martina

last: Leso

first: Salim

last: Hossain Reza

first: D


title: The role of Vitamin C and TET dioxygenases in genome-wide features of regulatory T cells [CMS-IP]

city: La Jolla

first: Anjana

last: Rao

address: 9420 Athena Cir

department: Signaling and Gene Expression

institute: La Jolla Institute for Allergy and Immunology

summary: TET enzymes are essential for the stability and function of regulatory T cells (Tregs), which maintain immune homeostasis and self-tolerance and express the lineage-determining transcription factor Foxp3. We previously showed that Vitamin C acts through TET enzymes to maintain the demethylated status of CNS2, a key intronic enhancer of the Foxp3 gene that is essential for the stability of Foxp3 expression both in vivo and in vitro. Here we show that Vitamin C enables genome-wide features of “induced” T regulatory cells (iTregs) in vitro that overlap with those induced by TET proteins during Treg differentiation in vivo. Vitamin C enhances IL-2 responsiveness in iTregs by increasing phospho-STAT5 levels, STAT5 occupancy and DNA demethylation at key Treg-specific enhancers, and maintains the stable expression of Treg-specific genes including Foxp3 and Il2ra. Our data will be relevant to future studies of the association between plasma Vitamin C levels, Treg function and autoimmunity in humans.

overall_design: RNA-seq, ATAC-seq, CMS-IP (5hmC mapping) and WGBS datasets

first: Xiaojing

last: Yue

first: Daniela

last: Samaniego Castruita

first: Benjamin

last: Barwick


title: The role of Vitamin C and TET dioxygenases in genome-wide features of regulatory T cells [ATAC-seq]


title: The role of Vitamin C and TET dioxygenases in genome-wide features of regulatory T cells [RNA-seq]


title: mRNA-sequencing of METTL3-deficient NK cells

status: Public on Jul 20 2021

city: Hefei

first: Zhigang

last: Tian

state: Anhui

address: Huangshan Road，number 443

country: China

institute: Univerity of Science and Technology of China

summary:
Purpose: For comparing the transcript changes, we conducted mRNA-sequencing of splenic NK cells from Ncr1Cre-Mettl3fl/fl (cKO) and Mettl3fl/fl (WT) mice.
Method: Firstly, The splenic NK cells (CD45.2+CD3-NK1.1+NKp46+) are purified via Fluorescence activated Cell Sorting (FACS), then frozen in -80 °C ultra-low temperature refrigerator, followed by High-throughput sequencing, in three replicates, using Illumina Hiseq 1500 platform.
Result: Using standard data process workflow, we  found the differentailly expressed genes in NK cells from Ncr1Cre-Mettl3fl/fl (cKO) mice, compared with those from Mettl3fl/fl (WT) mice.

overall_design: Find the DEGs in cKO NK cells.

first: Hao

last: Song


title: The role of Vitamin C and TET dioxygenases in genome-wide features of regulatory T cells [WGBS]

type: Methylation profiling by high throughput sequencing


title: PRC1 drives Polycomb-mediated gene repression by controlling transcription initiation and burst frequency [ChIP-seq]

city: Oxford

first: Paula

last: Dobrinić

address: South Parks Road

department: Department of Biochemistry

institute: University of Oxford

summary: The Polycomb repressive system plays a fundamental role in controlling gene expression during mammalian development. To achieve this, Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) bind target genes and use histone modification-dependent feedback mechanisms to form Polycomb chromatin domains and repress transcription. The interrelatedness of PRC1 and PRC2 activity at these sites has made it difficult to discover the specific components of Polycomb chromatin domains that drive gene repression and to understand mechanistically how this is achieved. Here, by exploiting rapid degron-based approaches and time-resolved genomics we kinetically dissect Polycomb-mediated repression and discover that PRC1 functions independently of PRC2 to counteract RNA polymerase II binding and transcription initiation. Using single-cell gene expression analysis, we reveal that PRC1 acts uniformly within the cell population, and that repression is achieved by controlling transcriptional burst frequency. These important new discoveries provide a mechanistic and conceptual framework for Polycomb-dependent transcriptional control.

sample_organism:
Drosophila melanogaster
Homo sapiens
Mus musculus

overall_design:
Mouse embryonic stem cells in which PRC1 can be depleted via the auxin-inducible degron (AID) system were profiled for genomic distribution of Polycomb factors (RING1B, SUZ12), histone modifications (H2AK119ub1, H3K27me3, H3K4me3) and RNA polymerase II (total Pol II, Ser5P-Pol II, Ser2P-Pol II) using spike-in calibrated ChIP-seq. Cells were treated with auxin for 2, 4, 8 or 24 hours in three independent biological replicates.
Another mouse ESC line in which PRC2 can be depleted via the dTAG-inducible degron system was profiled in three independent biological replicates for genomic distribution of H3K27me3 before and after 2 hours treatment with dTAG-13 compound. Additionally, RING1B binding was compared between wild type (TIR1) and PRC1deg cells before auxin treatment, and H3K27me3 enrichment was compared between wild type (E14) and PRC2deg cell before dTAG-13 treatment.

first: Robert

last: Klose


title: Mettl3-RIP-sequencing and m6A-MeRIP-sequencing of NK cells


title: PRC1 drives Polycomb-mediated gene repression by controlling transcription initiation and burst frequency [RNA-seq]

sample_organism:
Drosophila melanogaster
Mus musculus

platform_organism:
Drosophila melanogaster
Mus musculus

overall_design:
Mouse embryonic stem cells in which PRC1 can be depleted via the auxin-inducible degron (AID) system were profiled for gene expression using spike-in calibrated nuclear RNA-seq. Cells were treated with auxin for 2, 4, 8 or 24 hours in three independent biological replicates, and gene expression changes were analysed relative to the control untreated cells.
Another mouse ESC line in which PRC2 can be depleted via the dTAG-inducible degron system was profiled in a similar manner to compare gene expression before and after 2 hours treatment with dTAG-13 compound.


title: Time-course transcriptome profiling in vasopressin-treated mouse kidney collecting duct mpkCCD

city: Baltimore

first: Hyun Jun

last: Jung

address: 720 Rutland Ave, Ross Research Building 1165B

department: Medicine (Nephrology)

institute: Johns Hopkins University School of Medicine

summary:
Purpose:  The goal of this study is to identify vasopressin-regulated genes in mouse kidney collecting duct cell line mpkCCD. To explore dynamic regulation of vasoressin-mediated transcriptional regulation, transcriptome of vasopressin-treated cells at four different time points (3h, 6h, 12h, and 24h) were profiled and analyzed.
Methods: Total RNAs were isolated from vasopressin-treated mpkCCD cells at different time points (3h, 6h, 12h, and 24h). Three replicates for vehicle- or vasopressin-treated group were generated at each tested time point. cDNA libraries were prepared using a Nextera DNA library preparation protocol. The sequence reads from Illumina HiSeq3000 platform were qualified and quantified at the transcript level using Salmon (0.14.1). Differential expression analysis were performed using edgeR.
Results: mRNA profiles of mouse kidney collecting duct mpkCCD cells treated with vasopressin analog (dDAVP) for four different time potins (3h, 6h, 12h, and 24h) were generated using an optimized RNA-Seq workflow. Transcript level quantification using a pseudo-alignment quantification method (Salmon) was performed to calculate transcript abundance in each sample. Then differential expression analysis identified the differentially expressed genes including Aqp2 gene at each time point comparison (dDAVP vs vehicle, FDR < 0.05). In addition, several new vasopressin-responsive genes that have not been elucidated before were identified.
Conclusions: This study revealed dynamic changes of vasopressin-responsive gene expression within 24 hours in mouse kidney collecting duct cells. The results from time-course transcriptome profiling identified the known vasopressin-responsive genes and several novel gene that are regulated temporally.

overall_design: Time-course vasopressin response of transcriptome in mouse kidney collecting duct cell line mpkCCD

first: Lihe

last: Chen

first: Mark

middle: A

last: Knepper


title: The combined detection of Amphiregulin, Cyclin A1 and DDX20/Gemin3 expression predicts aggressive forms of Oral Squamous Cell Carcinoma

city: La Tronche

first: Sophie

last: Rousseaux

address: Bd de la Chantourne

department: IAB

country: France

institute: INSERM-UGA

summary: Large-scale genetic and epigenetic deregulations enable cancer cells to ectopically activate tissue-specific expression programs. A specifically designed strategy was applied to oral squamous cell carcinomas (OSCC) in order to detect ectopic gene activations and develop a prognostic stratification test.  A dedicated original prognosis biomarker discovery approach was implemented using genome-wide transcriptomic data of OSCC, including training and validation cohorts.  Abnormal expressions of silent genes were systematically detected, correlated with survival probabilities and evaluated as predictive biomarkers. The resulting stratification test was confirmed in a third cohort using immunohistochemistry.  In order to address the potential interest of the ectopic expressions as therapeutic targets, the transcriptome of OSCC cells in which one of the predictive marker genes, CCNA1, was inactivated was analysed by RNAseq.  The genome-wide expression profile of an OSCC cell line, where CCNA1 had been inactivated was found similar to that of aggressive tumours positive for the three genes.  The exploration of the whole gene expression profile characterizing aggressive OSCC tumours highlights their enhanced proliferative and poorly differentiated intrinsic nature. Our experimental targeting of CCNA1 in OSCC cells is associated with a shift of transcriptomic signature towards the less aggressive form of OSCC, suggesting it could be a good target for therapeutic approaches.

overall_design: Transcriptomic (RNAseq) analysis of the OSCC cell line FaDu (from ATCC) after CCNA1 knock-down and in control cells (triplicates)

first: Florent

last: Chuffart

first: Anne-Laure

last: Vitte

first: Saadi

last: Khochbin


title: m6A-MeRIP sequencing of NK cells

summary: Total RNAs were isolated from WT splenic NK cells, and subjected to standard m6A MeRIP, in two replicates, using Illumina Novaseq 6000 platform. The raw sequencing reads were mapped to the genome of Mus musculus (mm10) with default parameters. ExomePeak was used to identify m6A peaks, which were annotated by intersection with gene architecture using ChIPseeker. Sequence motifs enriched in peak regions were identified using Homer.

overall_design: m6A modified mRNAs from WT splenic NK cells were immunopriciptated and enriched with m6A specific antibody, followed by deep sequencing, in two replicates, using Illumina Novaseq 6000 platform.


title: The SWI/SNF complex regulates the expression of miR-222, a tumor suppressor microRNA in lung adenocarcinoma

city: Granada

first: Jordi

last: Martorell-Marugan

address: Avenida de la Ilustración 114

department: Bioinformatics Unit

institute: GENyO

type: Non-coding RNA profiling by high throughput sequencing

summary: Mammalian SWI/SNF complexes are considered as key epigenetic regulators and they are recurrently mutated in many types of cancer. Most of the studies of these chromatin remodeling complexes are focused on their role in regulating protein-coding genes. However, here we show that the SWI/SNF complex is able to control the expression of microRNAs. We used a SMARCA4-deficient model of lung adenocarcinoma where we could track changes of the miRNome upon SMARCA4 restoration. We found that exogenous SMARCA4 was successfully incorporated into endogenous SWI/SNF complexes and that these SMARCA4-SWI/SNF complexes induced significant changes in the expression of cancer-related microRNAs. The most significantly dysregulated miRNA was miR-222, whose expression was promoted by SMARCA4-SWI/SNF complexes but not by SMARCA2-SWI/SNF complexes via their direct binding to a miR-222 enhancer region. Importantly, miR-222 expression decreased cell viability and impaired cell clonogenicity, phenocopying the tumor-suppressor role of the SMARCA4-SWI/SNF complex in lung cancer. Finally, we showed that the miR-222 enhancer does not interact with any cancer-related protein-coding genes, supporting that its tumor suppressor effect may be exerted via miR-222. Overall, this study highlights the relevant role of the SWI/SNF complex in regulating the expression of the non-coding genome.

overall_design: A549 cell line was transfected with an empty vector and a vector for restoration of SMARCA4 expression. Three independent biological replicates were analysed.

first: Paola

last: Peinado

first: Alvaro

last: Andrades

last: Martorell-Marugán

first: Jeffrey

middle: R

last: Haswell

first: Frank

last: Slack

first: Pedro

last: Carmona-Sáez

last: Medina


title: Reconstruction of human alveoli in vivo using fiber-shaped expansion system of iPS cell-derived lung progenitor cells

city: Kyoto

first: Shimpei

last: Gotoh

address: 53 Kawaharacho, Shogoin, Sakyo-ku

department: Center for iPS cell Research and Application

institute: Kyoto University

summary: We transplanted human iPS cell-derived lung progenitors into the mouse lung. In addition, human iPS cell-derived lung progenitors were expanded in the cell-encapsulated alginate microfibers. In each case, the lung progenitors were differentiated into respiratory epithelial cells in vitro and in vivo, respectively.

overall_design: Lung progenitors were induced from human iPS cells and the engrafted cells were collected eight weeks after transplantation of lung progenitors into the mouse lung. On the other hand, human iPS cell-derived lung progenitors were expanded in the cell-encapsulated alginate microfibers (three-dimensional culture) for four weeks and differentiated into alveolar and airway epithelial cells for additional two weeks. The transcriptomes of those cells were obtained based on paired-end reads (100-bp) with Illumina Novaseq 6000.

first: Satoshi

last: Ikeo


title: Mettl3-RIP-sequencing of NK cells

first: Zhigang

last: Tian

summary: m6A modification plays vital roles in regulating mRNA lifecycle, thus controlling the biological process of multiple cell types. Here we intended to discover the binding sites of Mettl3 protein on mRNA of NK cells. Total RNAs were isolated from WT splenic NK cells, and subjected to standard RIP protocol. The library products corresponding to 200-500 bps were enriched, quantified and finally sequenced on Illumina Novaseq 6000 sequencer with PE150 model. Mettl3 binding sites were located primarily in the 3’ untranslated regions (UTRs), coding sequences (CDSs) region and near stop codons.

overall_design: Mettl3-RIP sequencing of WT splenic NK cells.

first: Hao

last: Song


title: Early reduction in PD-L1 expression predicts faster treatment response in human cutaneous leishmaniasis

city: York

first: Paul

last: Kaye

address: Wentworth way

institute: University of York

summary: Whole blood transcriptomes taken from patients with cutaneous leishmaniasis at presentation and during sodium stibogluconate treatment.

overall_design: Comparison of whole blood transcriptomes before and during drug treatment

first: Nidhi

last: Dey

first: Sujai

last: Senarathna

first: Vijani

last: Somaratne

first: Nayani

last: Madarasinghe

first: Bimalka

last: Seneviratne

first: Sarah

last: Forrester

first: Marcela

last: Montes De Oca

first: Luiza

last: Campos Reis

first: Srija

last: Moulik

first: Pegine

last: Walrad

first: Mitali

last: Chatterjee

first: Hiro

last: Goto

first: Renu

last: Wickremasinghe

first: Dimitris

last: Lagos

first: Shalindra

last: Ranasinghe


title: Using ATAC-Seq to investigate the impact of vaccination on the epigenome

city: Adelaide

first: Feargal

last: Ryan

address: North Terrace

institute: SAHMRI

summary: We used a mouse model to investigate whether live (BCG) and non-live (DTPw) vaccines differentially reprogram trained immune responses and to investigate whether immunisation with DTPw altered susceptibility to sepsis. We found that compared to BCG-immunised mice, DTPw immunisation led to altered monocyte and dendritic cell (DC) cytokine responses to subsequent secondary stimuli, as well as genome-wide changes to chromatin accessibility in DCs. These effects were evident months after immunisation and are consistent with different programs of trained immunity being induced by live versus non-live vaccines.

overall_design: There are 12 samples total from 12 individual mice at one timepoint. 5 with each vaccine (BCG and DTPw) and 2 not vaccinated which are referred to as mock. Dendritic cells from these were subjected to ATAC-Seq.

first: David

last: Lynn

first: Natalie

last: Stevens


title: Alternative 3’UTRs play a widespread role in translation-independent mRNA association with endoplasmic reticulum

city: Philadelphia

first: Bin

last: Tian

address: 3601 Spruce Street

department: Program in Gene Expression and Regulation; Center for Systems and Computational Biology

institute: Wistar Institute

summary: Transcripts encoding membrane and secreted proteins are known to undergo translation on endoplasmic reticulum (ER). Translation-independent ER association (TiERA) has been reported for certain mRNAs, but the phenomenon is poorly understood. Here, using cell fractionation, polysome profiling, and 3’ end sequencing, we examine TiERA of poly(A)+ RNAs in mouse C2C12 myoblast cells. We identify transcript features that facilitate TiERA, including transcript size and GG and GC content. Consistent with the feature analysis, alternative polyadenylation (APA) isoforms differ substantially in TiERA, with long 3’UTR isoforms generally having a higher TiERA potential than short 3’UTR isoforms. Importantly, the widespread 3’UTR lengthening taking place in cell differentiation leads to greater transcript association with ER in differentiated myotubes, despite that the TiERA potential being generally maintained. In addition, we show that TiERA correlates negatively with mRNA stability, highlighting 3’UTR-mediated mRNA decay on ER. Together, our data indicate that sequence and size features impact ER association independent of translation, leading to distinct mRNA metabolism for different transcript groups, and APA can alter transcript stability and translation through isoform-specific ER association.

overall_design: Three RNAseq samples and 26 3'READS+ samples.

first: Larry

last: Cheng

first: Dinghai

last: Zheng


title: RNA-seqs of SRSF3 and hnRNPK depleted C2C12 cells

city: Nagoya

first: Akio

last: Masuda

state: Aichi

address: 65 tsurumai-cho, Showa-ku

institute: Nagoya University Graduate School of Medicine

summary: Total RNA was extracted from C2C12 cells transfected with the indicated siRNA using TRIZOL (Thermo Fisher Scientific), and was further purified with Quick-RNA Miniprep Kit (Zymo research) according to the manufacturer’s instructions. The extracted RNA was subjected to RNA-seq at Macrogen, Japan. Briefly, a sequencing library was prepared using the TruSeq Stranded mRNA kit (Illumina), and the library was read on Illumina NovaSeq 6000 (150 bp paired-end reads).

first: Toshihiko

last: Kawachi


title: Expression data from murine wildtype and Sh3gl1-/- B cells

status: Public on Jul 19 2021

city: Belfast

first: Dessi

last: Malinova

address: 97 Lisburn Road

department: WWIEM

institute: Queen's University Belfast

summary: We show that antigen internalization depends both on constitutive, clathrin-mediated endocytosis and on antigen-induced, clathrin-independent endocytosis mediated by endophilin A2. Although endophilin A2-mediated endocytosis is dispensable for antigen presentation, it is selectively required for metabolic support of B cell proliferation, in part through regulation of iron uptake. Consequently, endophilin A2 deficient mice show defects in GC B cell responses and production of high-affinity IgG. The requirement for endophilin A2 highlights a unique importance of clathrin-independent intracellular trafficking in GC B cell clonal expansion and antibody responses

overall_design: Wildtype or Sh3gl1 knockout transitional and follicular B cells were sorted by flow cytometry and used for RNA isolation. Follicular B cells from both genotypes were also treated with CD40L for 14 hours and RNA isolated for comparison. Three biological replicates were included from littermates of appropriate genotype.

first: Dessislava

first: Pavel

last: Tolar